Exosome of mesenchymal stem cell and preparation method and application thereof

Abstract

This invention belongs to the field of pet dermatology treatment, specifically relating to exosomes from mesenchymal stem cells, their preparation method, and applications. The preparation method includes: isolation and expansion of mesenchymal stem cells, serum-free culture induction, conditioned medium pretreatment, and exosome enrichment and purification. The resulting exosomes have a main peak particle size of 30-150 nm and express CD9, CD63, CD81, and TSG101 marker proteins. The exosomes of this invention can be prepared into sprays, gels, creams, and other dosage forms for the treatment of pet atopic dermatitis, skin trauma, burns, and refractory ulcers. They offer advantages such as high safety, good stability, strong penetrability, and convenient administration, avoiding the immune rejection and tumorigenic risks associated with cell therapy.

Description

Technical Field

[0001] This invention belongs to the field of pet skin disease treatment, specifically relating to an exosome of mesenchymal stem cells, its preparation method, and its application. Background Technology

[0002] Pet skin diseases, such as bacterial / fungal infections, trauma, burns, atopic dermatitis, chronic ulcers, and autoimmune skin diseases (such as pemphigus), are common health problems that trouble pets and their owners. Current mainstream treatments rely on corticosteroids, antibiotics, and immunosuppressants. While these drugs can temporarily control symptoms, long-term use can easily lead to serious side effects such as drug resistance, liver and kidney toxicity, and endocrine disorders, and their efficacy is limited for many chronic and intractable skin diseases.

[0003] Mesenchymal stem cells (MSCs) are a group of cells with self-renewal and multi-directional differentiation potential. MSCs are characterized by low immunogenicity, multiple sources and pathways, multi-differentiation potential, and strong paracrine function, which can restore homeostasis of the body through systemic functional regulation. Several studies have explored the therapeutic effects of MSCs in diseases, including myocardial infarction, autoimmune diseases, diabetes, liver injury, and kidney injury. MSC therapy involves multiple pathways, including anti-inflammatory, anti-oxidative stress, anti-endoplasmic reticulum stress, activation of the immune system, and promotion of regeneration of damaged sites. As an emerging therapy, MSC therapy has shown potential in the treatment of pet skin diseases. However, cell therapy has some inherent challenges: (1) uncertain cell survival, homing, and colonization efficiency; (2) concerns about potential tumorigenicity; and (3) high requirements and high costs for cell transportation and storage. Recent studies have found that the therapeutic effect of MSCs is mainly attributed to its paracrine function, and exosomes are the most critical functional mediators. Exosomes are extracellular vesicles with a diameter of about 30-150 nm that carry bioactive substances such as proteins, lipids, mRNA, and miRNA from the cell of origin. They can mediate intercellular communication and play multiple functions such as promoting tissue repair, regulating immunity, anti-fibrosis, and anti-inflammation.

[0004] Currently, there are no mature technologies or patents for the specific use of feline or canine MSCs exosomes in the treatment of pet skin diseases. Therefore, developing a skin disease treatment product based on feline or canine MSCs exosomes has significant clinical value and market potential.

[0005] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0006] To overcome the problem that existing technologies lack exosomes derived from cats or dogs for the treatment of skin diseases, this invention provides a method for preparing exosomes from mesenchymal stem cells, comprising the following steps:

[0007] (1) Isolation and expansion of mesenchymal stem cells: Mesenchymal stem cells were isolated from pet tissues and primary culture and early expansion culture were carried out using serum-containing expansion medium; (2) Serum-free culture induction: After the mesenchymal stem cells fuse, the serum-containing expansion medium is discarded, the cells are washed and replaced with serum-free medium, the cells are cultured and the cell culture supernatant is collected as conditioned medium; (3) Conditioned culture medium pretreatment: The conditioned culture medium is centrifuged to gradually remove dead cells and cell debris, and the supernatant is collected; (4) Exosome enrichment and purification: The supernatant was polymerized with exosome concentrate, the exosome precipitate was collected by centrifugation and resuspended, and then purified by ultrafiltration through an exosome purification filter column. The purified exosome suspension was collected.

[0008] Preferably, in step (1), the pet tissue is one of the following: adipose tissue of a dog or cat, Wharton's jelly of the umbilical cord, or dental pulp tissue; And / or, the serum-containing amplification medium is supplemented with 10%~15% fetal bovine serum and 1% penicillin-streptomycin in the basal medium; the basal medium is DMEM / F12 or α-MEM.

[0009] Preferably, in step (2), when the fusion rate of mesenchymal stem cells reaches 70-80%, the serum-containing expansion medium is discarded, the cells are washed and replaced with serum-free medium, and cultured for another 24-72 hours. The cell culture supernatant is then collected as a conditioned medium. And / or, the serum-free culture medium contains bFGF and EGF cytokines.

[0010] Preferably, in step (3), the conditioned medium is centrifuged at 300×g for 10 min, 2000×g for 20 min and 10000×g for 30 min at 4°C to gradually remove dead cells and cell debris and collect the supernatant.

[0011] Preferably, in step (4), the method of polymer precipitation of the supernatant using exosome concentrate is as follows: mix the exosome concentrate with the supernatant at a volume ratio of 1:4-5, let it stand at 4-6℃ for 2-10h, and then collect the precipitate by centrifugation at 10000×g for 60-80min. And / or, the ultrafiltration purification method of the exosome purification filter column is: centrifugation at 3000×g for 10-15 min.

[0012] Based on the same technical concept, the present invention further provides an exosome of mesenchymal stem cells obtained by the above preparation method.

[0013] Based on the same technical concept, the present invention further provides the application of the exosomes of the mesenchymal stem cells in the preparation of drugs for treating pet skin diseases, wherein the pet skin diseases are one or more of the following: allergic skin diseases, skin trauma, burns, intractable ulcers, bacterial skin infections, fungal skin infections, autoimmune skin diseases, and hair regeneration disorders.

[0014] Preferably, the allergic skin disease is atopic dermatitis; the autoimmune skin disease is pemphigus foliaceus; and the refractory ulcer is a diabetic ulcer or a pressure ulcer.

[0015] Based on the same technical concept, the present invention further provides a drug for treating pet skin diseases, the drug comprising exosomes of the mesenchymal stem cells and a pharmaceutically acceptable carrier; the content of the exosomes of the mesenchymal stem cells is 0.1-10 mg based on total exosome protein. The pharmaceutically acceptable carrier includes, but is not limited to: physiological saline, PBS buffer, hyaluronic acid solution, hydrogel, liposome, or cream matrix.

[0016] Preferably, the dosage form of the drug is one of the following: spray, gel, cream, injection, drops, or sustained-release wound dressing.

[0017] The beneficial effects of this invention are as follows: 1. This invention uses cell-free exosomes as active substances, which completely avoids the potential risks of immune rejection, tumorigenesis and ectopic embolism in intact mesenchymal stem cell therapy. It is especially suitable for long-term and multiple-dose clinical scenarios in pets, and the safety is significantly improved.

[0018] 2. Exosomes can be cryopreserved for a long time at -80℃, with a long shelf life. Their composition, particle size and activity are easier to standardize and quality control, making them convenient for large-scale preparation and storage as ready-made products. This overcomes the shortcomings of high requirements for the transportation and storage of live cells and large batch differences.

[0019] 3. Exosomes have a particle size of only 30-150nm, making it easy to penetrate the skin's stratum corneum barrier and reach the deep dermal tissues. The microRNAs such as miR-21, miR-31, and miR-125b, as well as various growth factors and cytokines they carry, can precisely regulate the proliferation and migration of fibroblasts and keratinocytes, promote angiogenesis, and regulate the local immune microenvironment to achieve multi-target synergistic therapy.

[0020] 4. The exosomes of this invention can be prepared into various local drug delivery formulations such as sprays, gels, creams, and wound dressings. The drug delivery operation is simple and does not require professional medical personnel, which significantly improves the medication compliance of pet owners and is particularly suitable for home care and long-term management of chronic skin diseases.

[0021] 5. Exosomes can be used in combination with traditional drugs such as low-dose antibiotics and glucocorticoids to enhance efficacy while significantly reducing the dosage and frequency of administration of traditional drugs. This helps to reduce the risk of drug resistance and side effects such as liver and kidney toxicity and endocrine disorders, providing a new strategy for the treatment of refractory skin diseases in pets. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a graph showing the particle size and concentration of exosomes from adipose-derived mesenchymal stem cells.

[0024] Figure 2 This is a graph showing the particle size and concentration of exosomes from umbilical cord mesenchymal stem cells.

[0025] Figure 3 These are TEM images of adipose-derived mesenchymal stem cell exosomes (left) and umbilical cord mesenchymal stem cell exosomes (right).

[0026] Figure 4 This is a protein expression diagram of adipose-derived mesenchymal stem cell exosomes (left) and umbilical cord mesenchymal stem cell exosomes (right). Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0028] This invention provides a method for culturing exosomes of canine / cat mesenchymal stem cells, comprising the following steps: (I) Methods for the isolation and expansion of canine / cat mesenchymal stem cells: This section describes in detail the methods for isolating, primary culturing and expanding mesenchymal stem cells (MSCs) from autologous or allogeneic tissues of healthy dogs or cats. The preferred tissue sources are adipose tissue, Wharton's jelly from the umbilical cord or dental pulp. The entire operation must be carried out in a biosafety cabinet that meets GMP requirements, using sterile reagents and consumables.

[0029] (I-1) Culture and digestion solution (1) Amplification medium (containing serum): The basal medium (such as DMEM / F12, α-MEM) is supplemented with fetal bovine serum (FBS, preferably a qualified batch that has been tested for viruses and is free of mycoplasma contamination, with a final concentration of 10%-15%), and 1% penicillin-streptomycin (double antibody) is added. This medium is used for primary cell isolation and early amplification to promote cell adhesion and proliferation.

[0030] (2) Induction / maintenance medium (serum-free): Commercial serum-free mesenchymal stem cell culture medium (such as StemProMSC SFM, or self-prepared serum-free medium with added specific cytokines such as bFGF, EGF, etc.). This medium does not contain animal serum and is used for a specific stage after the cell fusion rate has reached the target, in order to reduce the potential interference of serum with subsequent experiments or treatments.

[0031] (3) Primary digestion solution: Type I collagenase (for adipose tissue and Wharton's gum) or Dispase enzyme (for dental pulp), with an activity ≥200U / mg, aseptically prepared in Hank's balanced salt solution (HBSS) to a final concentration of 0.1%-0.2%.

[0032] (4) Subdigestion solution: recombinant trypsin substitutes (such as TrypLE) TM Select (no animal / human-derived components, low cytotoxicity) or 0.25% trypsin-EDTA (serum-containing media require termination with trypsin inhibitors or serum-containing media).

[0033] (5) Washing solution: sterile phosphate buffer solution (PBS, pH 7.2-7.4).

[0034] (I-2) Tissue Collection Method: The tissue source for canine / feline MSCs in this section is as follows: tissues were collected under aseptic conditions from healthy adult or newborn dogs / cats after obtaining informed consent from the owner and approval from the institution's animal ethics committee; among which: Adipose tissue: Approximately 1-3g of adipose tissue was collected from the groin or interscapular fat pad.

[0035] Umbilical cord tissue: Collect approximately 5-10cm of umbilical cord from healthy, full-term newborn dogs / cats delivered by cesarean section or normal delivery.

[0036] Pulp tissue: The pulp separated from deciduous or extracted primary / permanent teeth.

[0037] The data collection and processing process is briefly described below: (1) Immediately after collection, place the tissue in sterile preservation solution (physiological saline or basal culture medium containing high concentration of double antibodies) and transport it to the GMP laboratory at low temperature (4°C) within 2-4 hours.

[0038] (2) After receiving the sample, the laboratory checks its appearance and whether it is contaminated, and registers the donor information (breed, age, gender, etc.).

[0039] (3) Organizational processing: (3-1) Fat: Remove visible blood vessels and fascia, wash thoroughly with PBS, and cut into 1mm pieces. 3 Fragments.

[0040] (3-2) Umbilical cord: Clean bloodstains with PBS, remove outer membrane and blood vessels, peel off Wharton's jelly, and cut into 1-3mm pieces. 3 Organizational block.

[0041] (3-3) Dental pulp: Split open the tooth, remove the dental pulp tissue with sterile forceps, wash with PBS, and cut into small pieces.

[0042] (4) Sampling was conducted for aerobic and anaerobic microbial culture and testing.

[0043] (5) Primary cell isolation: Enzymatic digestion (preferred) or tissue block adhesion method is used.

[0044] (5-1) Enzymatic digestion method: Mix the shredded tissue with the primary digestion solution and digest in a 37°C water bath with shaking for 30-90 min (the digestion time for adipose tissue can be extended to 1-2 hours). Add an equal volume of amplification culture medium (containing serum) to terminate digestion, filter through a 100μm cell sieve, and collect the cell pellet by centrifugation (300g, 5 min).

[0045] (5-2) Tissue block adhesion method: Spread the cleaned tissue block evenly on the bottom of the culture bottle, invert the culture bottle, add a small amount of amplification culture medium, and place it in a 37℃, 5% CO2 incubator for 2-4 hours. After the tissue block adheres, carefully turn the culture bottle over, add more culture medium and continue culturing.

[0046] (6) Place the obtained primary cells or adherent tissue blocks in amplification medium (containing serum) and culture them in a 37°C, 5% CO2, saturated humidity incubator. Change half of the medium every 3-4 days.

[0047] (I-3) Stem cell culture and medium change (1) Primary and early amplification culture (using serum-containing amplification medium) After primary cell seeding, cells were cultured in an expansion medium (containing serum). This medium contained fetal bovine serum, providing growth factors, hormones, and adhesion proteins necessary for cell adhesion, extension, and proliferation. Cell morphology and confluence were observed every 2-3 days.

[0048] (2) Change the medium after the fusion rate reaches the standard (use serum-free medium). When the cell monolayer growth observed under a microscope reaches 70%-80% confluence (the percentage of cell coverage of the bottom area of ​​the culture container), the following steps are performed: (2-1) Discard the original culture medium: Carefully aspirate or pour out all the original culture medium from the culture flask / vessel.

[0049] Note: In this step, "original culture medium" specifically refers to the "amplification culture medium (containing serum)," which is a complete culture medium supplemented with fetal bovine serum. At this point, the cells have entered the late logarithmic growth phase and are close to confluence. Certain unknown components or inhibitory factors in the serum may begin to affect the cell state, or it may need to be removed to meet the requirements of subsequent experiments (such as differentiation induction, gene expression analysis, and clinical-grade cell preparation) that require a serum-free environment.

[0050] (2-2) Washing cells: Add an appropriate amount of sterile PBS (about 5 mL, based on a T75 culture flask), gently shake to wash the cell layer 1-2 times to completely remove residual serum components (proteins in serum may interfere with subsequent serum-free culture), and discard the PBS.

[0051] (2-3) Replace with serum-free medium: Add induction / maintenance medium (serum-free) pre-equilibrated to 37°C, with the same volume as normal culture.

[0052] Note: This medium change does not constitute cell passage. The cells remain adherent and will continue to grow or undergo further treatment after the medium change. If passage is required, cells should be cultured in serum-free medium for a certain period of time, or cells at 70%-80% confluence should be directly passaged using standard digestion methods (during passage, you can choose to continue using serum-containing medium or transfer to a serum-free system, depending on specific needs).

[0053] (4) Continue culturing: Place the culture bottle back into the 37℃, 5% CO2 incubator for continued culturing.

[0054] (5) After culturing for 24-72 hours, collect the cell culture supernatant, which is the conditioned medium.

[0055] (II) Isolation and purification of exosomes (II-1) First, the conditioned medium is pretreated: under 4°C, centrifugation is performed sequentially at 300×g, 2000×g, and 10000×g to gradually remove large particulate impurities such as dead cells and cell debris.

[0056] (II-2) After completing the above pretreatment, collect the supernatant. Exosomes can be enriched using one or more combinations of ultracentrifugation, size exclusion chromatography, or polymer precipitation. The preferred method is to prepare the exosomes using a self-developed exosome extraction and purification kit combined with ultrafiltration. This kit includes an exosome concentration solution (ECS) and an exosome purification filter (EPF column), eliminating the need for ultracentrifugation equipment, simplifying the operation, and achieving high exosome purity. The specific steps are as follows: (1) Sample pretreatment (steps included with the kit) Although the present invention has performed stepwise centrifugation to remove large particles from the conditioned medium, for optimal operating procedures to be compatible with the kit, it is recommended that the collected supernatant be pretreated again as follows when using the kit: (1-1) Sample volume: Take 20 mL of cell culture supernatant after the above-mentioned pretreatment steps (minimum volume for single extraction, which can be scaled up proportionally).

[0057] (1-2) Centrifugation to remove debris: Transfer the sample to a 50 mL centrifuge tube and centrifuge at 3000 × g for 10 min at 4 °C to remove residual cell debris or precipitate. If there is a lot of precipitate, the centrifugation can be repeated once, and the supernatant can be collected each time.

[0058] (1-3) Transfer of supernatant: Carefully transfer the supernatant after centrifugation to a new 50mL centrifuge tube and record the volume.

[0059] (2) Exosome enrichment (polymer precipitation method) (2-1) Adding ECS ​​reagent: Add Exosome Concentration Solution to the supernatant at a ratio of 5 mL ECS reagent per 20 mL of cell supernatant. If the sample volume is not 20 mL, adjust the amount added proportionally.

[0060] (2-2) Mixing and standing: Tightly cap the centrifuge tubes and mix thoroughly using a vortex mixer for 1 min. Then place the centrifuge tubes in a 4°C refrigerator and let them stand for at least 2 hours. Extending the standing time can increase the exosome yield, but should not exceed 24 hours.

[0061] (2-3) Precipitation of exosomes: Centrifuge the mixture after standing at 4°C at 10000×g for 60 min. Discard the supernatant after centrifugation; the precipitate at the bottom of the tube is rich in exosome particles. Be careful to remove as much supernatant as possible to avoid loss of precipitate.

[0062] (2-4) Resuspension of exocrine cells: Add 1×PBS buffer (sterile) to the pellet. The dosage is 0.2 mL PBS for every 20 mL of initial cell supernatant. Gently pipette the pellet to dissolve it completely, and transfer the resuspension to a new 1.5 mL centrifuge tube.

[0063] (2-5) Harvesting crude exosomes: Centrifuge a 1.5 mL centrifuge tube containing the resuspension at 4°C at 12000×g for 2 min. After centrifugation, retain the supernatant, which is the crude extract rich in exosomes. If there is a lot of precipitation, repeat the centrifugation (12000×g / 2 min) until there is no obvious precipitation, and collect the supernatant each time.

[0064] (3) Exosome purification (ultrafiltration column method) (3-1) Sample loading: Transfer all the crude exosome extract obtained above into the upper chamber of the Exosome Purification Filter (EPF column). Note that the EPF column is for single use only and cannot be reused.

[0065] (3-2) Centrifugal purification: Place the EPF column into the matching collection tube and centrifuge at 3000×g for 10 min at 4℃.

[0066] (3-3) Collection and purification of exosomes: After centrifugation, discard the EPF column and collect the liquid at the bottom of the tube, which is the purified exosome particle suspension. This liquid is colorless and transparent and can be used directly for subsequent analysis or experiments.

[0067] (3-4) Storage: Aliquot the purified exosomes into sterile low-adsorption centrifuge tubes at 50-100 μL each, label them, and store them at -80℃ for long-term storage, avoiding repeated freeze-thaw cycles. For short-term storage (within 1 week), store at -20℃.

[0068] It should be noted that the above-mentioned reagent kit combined with ultrafiltration method used in this invention has the following advantages compared with the traditional ultracentrifugation method (100,000 × g, 70-90 min): (i) Low equipment requirements: No ultracentrifuge is required; a regular high-speed centrifuge (up to 10,000 x g) is sufficient.

[0069] (ii) Easy to operate: It takes only about 3-4 hours in total (including settling time) and does not require gradient density preparation.

[0070] (iii) High purity: Exosomes are selectively precipitated using ECS ​​reagents and large vesicles and protein aggregates are removed using EPF columns. The resulting exosomes have a main peak particle size in the range of 30-150 nm, which is consistent with the typical characteristics of exosomes and is suitable for electron microscopy, NTA, nucleic acid / protein analysis and cell / animal experiments.

[0071] The purified exosomes were resuspended in sterile PBS or physiological saline, and protein quantification was performed using the BCA method. The purified exosomes were then aliquoted and stored at -80°C.

[0072] (III) Identification of exosomes The particle size distribution and concentration of exosomes were detected using nanoparticle tracking analysis, confirming that their main peak was between 30-150 nm. Their typical "cup-shaped" or "biconcave disc-shaped" morphology was observed using transmission electron microscopy (TEM). Specific marker proteins, such as CD9, CD63, CD81, and TSG101, were detected using Western blotting, and the negative marker Calnexin should not be expressed.

[0073] Example 1 This embodiment provides a method for preparing canine adipose tissue and umbilical cord MSCs exosomes, the preparation method being as follows: (1) P3-P5 generation canine adipose mesenchymal stem cells and umbilical cord mesenchymal stem cells were seeded into T75 culture flasks and cultured in DMEM / F12 expansion medium containing 10% fetal bovine serum at 37°C, 5% CO2, and saturated humidity incubator. (2) When the cell fusion reaches 80%, discard the original serum-containing culture medium, gently wash the cell layer twice with sterile PBS to remove residual serum, and replace it with serum-free mesenchymal stem cell culture medium preheated to 37°C. (3) After culturing for another 48 hours, collect the supernatant of each cell culture, which is the conditioned medium; (4) The conditioned culture was subjected to differential centrifugation pretreatment at 4℃: centrifugation at 300×g for 10min to remove live cells, centrifugation at 2000×g for 20min to remove dead cells and debris, and centrifugation at 10000×g for 30min to remove large vesicles and organelle debris. The supernatant was collected after each centrifugation. (5) Take the supernatant after pretreatment and use the exosome extraction and purification kit combined with ultrafiltration to separate and purify exosomes: add exosome concentrate at a volume ratio of 4:1, mix well, let stand at 4℃ for 2h, centrifuge at 10000×g for 60min to collect the precipitate, resuspend in PBS, and centrifuge at 3000×g for 10min through a purification filter column to obtain purified exosomes. (6) Finally, the exosomes were suspended in 1 mL of sterile PBS and the protein concentration was determined by the BCA method: 3.15 mg / mL for lipid-derived exosomes and 2.19 mg / mL for umbilical cord-derived exosomes.

[0074] Example 2 This embodiment provides a method for preparing feline adipose-derived MSCs exosomes, the preparation method being as follows: (1) Take P4 generation cat adipose mesenchymal stem cells, seed them in T75 culture flasks, use DMEM / F12 expansion medium containing 10% fetal bovine serum, and culture them routinely in a 37℃, 5% CO2 incubator; (2) When the cell fusion reaches 75%, discard the original culture medium, wash the cell layer twice with sterile PBS, and replace it with serum-free mesenchymal stem cell culture medium. (3) After culturing for another 48 hours, the cell culture supernatant was collected as a conditioned medium; (4) The conditioned medium was pretreated by differential centrifugation at 4℃: centrifuged at 300×g for 10 min, 2000×g for 20 min, and 10000×g for 30 min in sequence. After each centrifugation, the supernatant was taken and cells and debris were removed. (5) Use an exosome extraction and purification kit combined with ultrafiltration to separate and purify exosomes: Add 1 / 4 volume of exosome concentrate to the supernatant, mix thoroughly, let stand at 4℃ for 3h, centrifuge at 10000×g for 60min and discard the supernatant. Resuspend the precipitate in sterile PBS, transfer it to a purification filter column, centrifuge at 3000×g for 10min, and collect the purified exosome suspension at the bottom of the column. (6) The purified exosomes were resuspended in 0.5 mL of sterile PBS, and the protein concentration was measured to be 2.68 mg / mL by BCA method.

[0075] Test Example 1 The canine adipose tissue and umbilical cord MSCs exosomes prepared in Example 1 were analyzed by NTA, and the results showed that: The concentration of exosome granules in adipose-derived mesenchymal stem cells was 8.84 × 10⁻⁶. 10 The particle size was 89.1 nm (e.g., 1 / mL) and the average particle size was 89.1 nm. Figure 1 As shown in the figure, the concentration of exosome granules in umbilical cord mesenchymal stem cells was 8.22 × 10⁻⁶. 10 The particle size is 88.0 nm (e.g., 1 / mL) and the average particle size is 88.0 nm. Figure 2 (As shown).

[0076] Figure 3 These are transmission electron microscopy (TEM) images, which demonstrate that each exosome sample has a lipid bilayer structure or cup morphology, with a diameter of approximately 30-150 nm.

[0077] Figure 4 The WB test results showed positive for CD63 and TSG101.

[0078] Test Example 2 In the "isolation and purification of exosomes" step of this invention, a systematic comparative experiment was conducted on the centrifugation conditions for "pretreatment with conditioned medium". The experimental setup is as follows: The first group involved centrifugation at 300×g only: an equal volume of conditioning culture medium was taken and centrifuged at 300×g for 10 min at 4℃. The supernatant was collected and used directly for subsequent exosome purification (such as ultracentrifugation or reagent kit precipitation).

[0079] The second group involved centrifugation at 300×g and 2000×g in sequence: take an equal amount of conditioning culture medium, centrifuge at 300×g for 10 min, take the supernatant and then centrifuge at 2000×g for 20 min, and collect the supernatant for subsequent exosome purification.

[0080] The third group involved centrifugation at 300×g, 2000×g, 10000×g, and 30000×g in sequence: Based on the second group, centrifugation was continued at 10000×g for 30 min, followed by centrifugation at 30000×g for 60 min. The final supernatant was collected for subsequent exosome purification.

[0081] The supernatants from the three pretreated groups were subjected to the same exosome extraction method (e.g., ultracentrifugation or precipitation) to obtain exosome precipitates. Particle concentration and size distribution were detected by nanoparticle tracking analysis (NTA), and exosome markers (CD9, CD63, CD81) and cell debris-related proteins (e.g., cytochrome C) were detected by Western blot. The results are compared with those of this invention in Table 1.

[0082] Table 1

[0083] As shown in Table 1: (i) When centrifuged at 300×g, a large number of cell debris and microvesicles remained in the supernatant. The final exosome product contained as many as 42.3% large particles >200nm, which was seriously contaminated and had the worst effect. (ii) After centrifugation at 2000×g, the proportion of large particles decreased to 18.7%, and the contamination was significantly improved. However, some larger vesicles were still not removed, which affected the purity of subsequent exosomes. (iii) When centrifugation is further increased to higher intensities such as 30,000×g, the proportion of particles >200nm only decreases from 18.7% to 12.5%, which is a limited improvement (only a decrease of about 6 percentage points). Moreover, the additional high centrifugal force may lead to exosome loss (recovery rate decreases by about 15%-20%), while significantly prolonging the processing time. The economy and operability are not ideal. The three-step centrifugation scheme of 300×g, 2000×g and 10000×g adopted in this invention can control the proportion of large particles >200nm to about 13.8% (comparable to the 30000×g group), while ensuring the exosome recovery rate (≥85%), and without the need for ultra-high speed centrifugation equipment, achieving the best balance between removal efficiency, sample recovery rate and process simplicity.

[0084] Verification Example 3: Exosome hydrogels promote skin wound healing in dogs (a) Preparation of hydrogels containing exosomes The exosomes (total protein content 100 μg) obtained in Example 1 were mixed evenly with 1% hyaluronic acid hydrogel.

[0085] (II) Establishing a full-thickness skin defect trauma model in dogs (1) Experimental animals: Six healthy adult beagles (half male and half female, weighing 10-15 kg) were provided by the experimental animal center and were acclimatized for one week before the experiment. The experimental process followed animal welfare and the "3R" principle.

[0086] (2) Modeling method: (2-1) Preoperative preparation: Experimental dogs were fasted for 12 hours and deprived of water for 4 hours. Propofol (4-6 mg / kg) was administered via intramuscular injection for anesthesia (isoflurane inhalation was used for maintenance if necessary). After successful anesthesia, the dogs were fixed in a prone position on the operating table.

[0087] (2-2) Hair removal and disinfection: Use electric clippers to shave the hair from the back of the dog below the shoulder blades to the lumbosacral region, apply hair removal cream for further treatment, disinfect with iodine, and cover with a sterile drape.

[0088] (2-3) Wound preparation: On the midline of the dog's back, below the scapula, use a sterile scalpel and ophthalmic scissors to remove three 4cm × 4cm square pieces of full-thickness skin tissue (including epidermis, dermis, and subcutaneous tissue) along the marked line (spaced ≥ 3 cm apart to avoid mutual interference), down to the fascia layer, avoiding damage to the muscle layer. Bleeding from the wound is controlled by applying pressure with sterile gauze.

[0089] (2-4) Randomization and intervention: Each dog's three wounds were randomly assigned to Group A (exosome hydrogel), Group B (blank hydrogel), and Group C (blank control, covered only with sterile dressing) according to a random number table. The corresponding preparation was applied immediately after modeling and changed every 2 days for 14 consecutive days. The application of the preparation and wound measurement were performed by different personnel, and the measurement personnel kept the grouping blinded.

[0090] (2-5) Wound protection: After covering the wound with the preparation, the entire wound is secured with sterile gauze and an elastic bandage in a figure-eight pattern. An Elizabethan collar is worn postoperatively to prevent licking, scratching, and dressing dislodgement. Meloxicam (0.2 mg / kg, subcutaneous injection, once daily for 3 days) is administered postoperatively for pain relief.

[0091] (2-6) Model verification: After modeling, the wound was observed to be bright red, with visible subcutaneous fat layer and myofascia, and no skin residue, confirming the successful establishment of the full-thickness skin defect model. Postoperatively, the dog's diet and mental state were normal, and no obvious systemic or local inflammatory reactions such as redness, swelling, heat, and pain were observed.

[0092] (III) The experiment was divided into three groups. A self-controlled design was used, with n=6 wounds in each of the three groups A, B, and C (from the same 6 dogs), and the individual themselves served as the control to eliminate individual differences.

[0093] (iv) Observation indicators and detection methods (4-1) Wound area measurement and wound contraction rate calculation Measurement method: Digital photographs of the wound were taken under anesthesia on postoperative day 0 (the day of modeling), day 7, and day 14 (with fixed camera height and angle, and a ruler placed next to the wound). The wound outline was delineated and the wound area was calculated using ImageJ image analysis software.

[0094] Formula for calculating wound contraction rate: Wound contraction rate = (S0) / (S0) S t ) / S0×100% in: S0 = Initial wound area (day 0 post-surgery); S t =Wound area on postoperative day t; Unit: cm 2 .

[0095] (4-2) Histological observation On postoperative day 7, under sedation, a small amount of tissue was harvested from the wound margin using a 3 mm skin biopsy trephine. On postoperative day 14 (after the final area measurement), tissue was harvested from the center and margins of the wound using a 6 mm biopsy trephine. The harvested tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm sections for HE and Masson staining to observe granulation tissue growth, collagen deposition, and epithelialization. The wounds were routinely dressed and cared for until healing. After the experiment, the dogs were kept and observed at the experimental animal center.

[0096] (v) Results On postoperative days 7 and 14, the wound contraction rate in group A was significantly higher than that in groups B and C (p<0.01), granulation tissue growth was more vigorous, and epithelialization process was faster.

[0097] (5-1) Wound contraction rate (key indicator) The results of the wound shrinkage rate measurement are shown in Table 2: Table 2 Comparison of wound area closure rate among groups (n=6)

[0098] Note: Linear mixed model (LMM) analysis was used, and Bonferroni correction was applied for post-hoc tests. Different letters in the same column (a, b, c) indicate statistically significant differences between groups (P < 0.05). There were no statistically significant differences in the initial area (S0) among the three groups (P > 0.05), indicating comparability.

[0099] (5-2) Observation of wound healing Day 7 post-surgery: Group A: The wound area has significantly shrunk, and a large amount of new granulation tissue can be seen at the edge of the wound. It is bright red, granular, soft in texture, and bleeds easily when touched. Epithelial cells can be seen migrating from the edge to the center at the bottom of the wound, and the epithelialization process is obvious.

[0100] Group B: The wound shrinkage was moderate, and the granulation tissue growth was sparser and lighter in color than that of Group A.

[0101] Group C: The wound did not shrink significantly, a small amount of yellow exudate was visible on the wound, granulation tissue grew slowly, and epithelialization at the wound edge was not obvious.

[0102] Day 14 post-surgery: Group A: The wound has basically healed, the wound area has shrunk by about 78.6%, new epithelium is visible on the wound surface, the wound is smooth, and no obvious scar hyperplasia is observed.

[0103] Group B: The wound area shrank by approximately 58.4%, but unhealed areas were still visible in the center of the wound, and the granulation tissue filling was incomplete.

[0104] Group C: The wound area shrank by about 45.2%, but there was still a large defect in the center of the wound, and the healing process was significantly delayed.

[0105] (5-3) Histopathological results HE staining (day 7 post-surgery): Group A: The dermis shows a large number of newly formed capillaries and fibroblasts, with less inflammatory cell infiltration and high maturity of granulation tissue; the epidermis shows epithelial cells extending in a tongue shape towards the center of the wound.

[0106] Group B: Fewer newly formed capillaries and fibroblasts than Group A, and more inflammatory cell infiltration.

[0107] Group C: Sparse granulation tissue, mainly infiltrated by inflammatory cells, with disordered arrangement of fibroblasts.

[0108] HE staining (day 14 post-surgery): Group A: The wound is basically covered by new epidermis, the epidermis is thickened, the dermal collagen fibers are arranged in an orderly manner, and skin appendages (hair follicles, sebaceous glands) begin to regenerate.

[0109] Group B: The wound was partially covered by the epidermis, and the collagen fibers were arranged in a more disordered manner.

[0110] Group C: The wound still has significant defects, little collagen deposition, and incomplete epidermal coverage.

[0111] Masson staining (day 14 post-surgery): Group A: Collagen fiber deposition is abundant, blue-green in color, densely and orderly arranged, and the ratio of type I / III collagen tends to be normal.

[0112] Group B: Collagen fiber deposition is moderate and the arrangement is relatively loose.

[0113] Group C: Collagen fiber deposition is sparse and disordered.

[0114] Verification Example 4: Exosome Spray Treatment for Feline Atopic Dermatitis (a) Laboratory animals Three domestic cats (three Chinese domestic cats, weighing 3.5-5.2 kg and aged 2-4 years) clinically diagnosed with feline atopic dermatitis (FAS) were selected. All of them presented with multiple erythema, hair loss and itching on their backs.

[0115] (II) Diagnostic Criteria Meeting the Favrot criteria includes: i) a history of chronic or recurrent pruritus; ii) the main lesions are located on the back or waist; iii) exclusion of other causes such as parasitic infections, fungal infections, and food allergies; and iv) a response to glucocorticoid treatment.

[0116] (III) Quantitative assessment of itching behavior The Dual-Dimensional Visual Analogue Scale for Cat Itching (VAScat) was used. The pruritus behavior of cats was quantitatively assessed. The scale consists of two independent dimensions: licking and scratching. Each dimension uses a visual analog scale (VAS) score from 0 to 10 cm. The highest score (VAS-max) is taken as the final pruritus score. The judgment criteria are shown in Table 3.

[0117] Table 3 Criteria for Judging Itching Behavior

[0118] Method for counting scratching frequency: Before treatment and on days 7 and 14 after treatment, the same observer conducted quantitative statistics using a 30-minute video recording method. The affected cat was placed in an observation cage, and video was recorded continuously for 30 minutes, recording the frequency of the following behaviors: Number of licks: The number of times the tongue touches the skin (each consecutive lick counts as 1 lick). Number of scratches: The number of times the hind limbs scratch the skin (each consecutive scratch is counted as 1 time). VAS-max score: The score is assigned by the pet owner on the VAScat scale based on the average licking / scratching frequency over 24 hours. (iv) Method for measuring erythema area The area of ​​erythema was quantitatively measured using ImageJ image analysis software combined with the SCORFAD scoring system. (4-1) Measurement steps: (i) Under standard lighting conditions (fixed light source angle and intensity), take digital photos of the affected area on the back of the cat and place a ruler next to the cat as a reference; (ii) Use ImageJ software to outline the erythema region and calculate the erythema area (unit: cm²). 2 ); (4-2) Observation time points: Photographic measurements were taken before treatment (baseline, Day 0) and on days 7 and 14 of treatment. The self-defined SCORFAD area grading (0-4 points) in the original manuscript was removed; all efficacy evaluations were based on the precise area (cm²) calculated using ImageJ. 2 As the sole quantitative indicator, it avoids the loss of statistical effectiveness caused by downgrading precise data to coarse-grained levels.

[0119] (v) Dosing regimen The exosomes (total protein content 100 μg / time) prepared in Example 2 were dissolved in 2 mL of physiological saline and loaded into a sterile spray device. The solution was sprayed onto the affected area once daily for two consecutive weeks.

[0120] Experimental design (self-comparison before and after): Treatment group (3 animals): received exosome spray therapy.

[0121] (vi) Experimental Results The preliminary experimental results show that exosome spray exhibits a preliminary trend towards therapeutic efficacy and good safety in this small sample (n=3), as detailed below: (6-1) Itching behavior shows a trend of improvement: Compared with the baseline before treatment (Day 0), the VAS-max score decreased by an average of 38.9% on day 7 of treatment (self-comparison, p<0.05); and by an average of 68.5% on day 14 of treatment (self-comparison, p<0.01).

[0122] The number of scratches per 30 minutes decreased by an average of 83.7% from baseline on day 14 of treatment.

[0123] (6-2) The area of ​​erythema showed a significant decreasing trend: Before treatment (Day 0), the average erythema area in the three affected cats was 4.5 cm². 2 (Range: 3.2-5.8cm)2 On the 14th day of treatment, the average affected area decreased to 1.4 cm. 2 (Range: 0.8-2.1cm) 2 ).

[0124] Before-and-after comparisons showed that the average reduction rate of erythema area reached 70.4% (paired t-test, p<0.05), suggesting that exosomes have a potential role in promoting the regression of this skin lesion.

[0125] (6-3) Secondary observation indicators: On the 14th day of treatment, fine, soft new hairs were observed to sprout on the affected areas of both cats.

[0126] (6-4) Safety observation: After 14 days of continuous use of the exosome spray, no adverse reactions such as local redness, increased desquamation, or systemic allergic reactions were observed in any of the three cats, suggesting that the preparation is well tolerated in topical application in cats.

[0127] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing exosomes from mesenchymal stem cells, characterized in that, The preparation method includes the following steps: (1) Isolation and expansion of mesenchymal stem cells: Mesenchymal stem cells were isolated from pet tissues and primary culture and early expansion culture were carried out using serum-containing expansion medium; (2) Serum-free culture induction: After the mesenchymal stem cells fuse, the serum-containing expansion medium is discarded, the cells are washed and replaced with serum-free medium, the cells are cultured and the cell culture supernatant is collected as conditioned medium; (3) Conditioned culture medium pretreatment: The conditioned culture medium is centrifuged to gradually remove dead cells and cell debris, and the supernatant is collected; (4) Exosome enrichment and purification: The supernatant was polymerized with exosome concentrate, the exosome precipitate was collected by centrifugation and resuspended, and then purified by ultrafiltration through an exosome purification filter column. The purified exosome suspension was collected.

2. The method for preparing exosomes from mesenchymal stem cells according to claim 1, characterized in that, In step (1), the pet tissue is one of the following: adipose tissue of a dog or cat, Wharton's jelly of the umbilical cord, or dental pulp tissue; And / or, the serum-containing amplification medium is supplemented with 10%-15% fetal bovine serum and 1% penicillin-streptomycin in the basal medium; the basal medium is DMEM / F12 or α-MEM.

3. The method for preparing exosomes from mesenchymal stem cells according to claim 1, characterized in that, In step (2), when the fusion rate of mesenchymal stem cells reaches 70-80%, the serum-containing expansion medium is discarded, the cells are washed and replaced with serum-free medium, and cultured for another 24-72 hours. The cell culture supernatant is collected as a conditioned medium. And / or, the serum-free culture medium contains bFGF and EGF cytokines.

4. The method for preparing exosomes from mesenchymal stem cells according to claim 1, characterized in that, In step (3), the conditioned medium is centrifuged at 300×g for 10 min, 2000×g for 20 min and 10000×g for 30 min at 4℃ to gradually remove dead cells and cell debris and collect the supernatant.

5. The method for preparing exosomes from mesenchymal stem cells according to claim 1, characterized in that, In step (4), the method of polymer precipitation of the supernatant by exosome concentrate is as follows: mix the exosome concentrate with the supernatant at a volume ratio of 1:4-5, let it stand at 4-6℃ for 2-10h, and then collect the precipitate by centrifugation at 10000×g for 60-80min. And / or, the ultrafiltration purification method of the exosome purification filter column is: centrifugation at 3000×g for 10-15 min.

6. Exosomes of mesenchymal stem cells obtained by the preparation method according to any one of claims 1-5.

7. The use of the exosomes of mesenchymal stem cells as described in claim 6 in the preparation of a drug for treating pet skin diseases, characterized in that... The pet skin diseases mentioned are one or more of the following: allergic skin diseases, skin trauma, burns, intractable ulcers, bacterial skin infections, fungal skin infections, autoimmune skin diseases, and hair regeneration disorders.

8. The application according to claim 7, characterized in that, The allergic skin disease is atopic dermatitis; the autoimmune skin disease is pemphigus foliaceus; the refractory ulcer is diabetic ulcer or pressure ulcer.

9. A drug for treating pet skin diseases, characterized in that, The drug comprises the exosomes of the mesenchymal stem cells as described in claim 6 and a pharmaceutically acceptable carrier; the content of the exosomes of the mesenchymal stem cells is 0.1-10 mg based on total exosome protein.

10. The medicament for treating pet skin diseases according to claim 9, characterized in that, The dosage form of the drug is one of the following: spray, gel, cream, injection, drops, or sustained-release wound dressing.

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